Anti-avian influenza virus antibody and application thereof

Abstract

The invention discloses an anti-avian influenza virus antibody and application thereof, and relates to the field of antibodies. The anti-avian influenza virus antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for detection of avian influenza virus, and has good activity, detection sensitivity and specificity.

Description

[0001] Cross - reference to related applications

[0002] This disclosure claims the priority of a Chinese patent application with the application number 202411633035.9 and the title "An Antibody Against Avian Influenza Virus and Its Application", filed with the Chinese Patent Office on November 14, 2024, the entire content of which is incorporated herein by reference. Technical field

[0003] The present invention relates to the technical field of antibodies, and more particularly, to an antibody against avian influenza virus and its application. Background art

[0004] Influenza virus, as a pathogen widely existing in nature, poses a serious threat to the health of humans and various animals. Among them, influenza A virus has become a key concern in the global public health field due to the variability of its antigens and high infectivity. Influenza A virus can be further divided into multiple subtypes, such as H1N1, H3N2, H5N1, H7N9, etc. These subtypes of viruses have differences in antigen structure, but they can all cause severe respiratory infections and even lead to pneumonia and death.

[0005] Avian influenza virus (H5N1), as a highly pathogenic influenza virus, poses a major threat to humans and poultry. In recent years, outbreaks of H5N1 subtype avian influenza virus have occurred continuously, attracting extensive attention to its transmission ability and potential public health impact. Its potential for recombination and mutation makes this virus a potential risk source for future influenza pandemics. Therefore, it is particularly important to develop detection and identification technologies for H5N1 virus.

[0006] The structure of H5N1 virus is complex, mainly composed of two glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA protein is the main surface antigen of the virus and plays a key role in the process of the virus invading host cells. The HA protein is like the "key" in the hands of the virus, binding to the receptor on the surface of the host cell through a specific receptor - binding site, thereby opening and invading the host cell. This unique binding mechanism makes the HA protein an ideal target for developing antibody - based detection technologies.

[0007] The immunoassay technology based on the HA protein can not only be used to detect the presence of H5N1 virus, but also provide important information for virus prevention and control and vaccine development by differentiating different variants of the virus. Traditional detection methods, such as virus isolation and PCR amplification, although having high sensitivity, are complex in operation, time-consuming, and require professional laboratory equipment and personnel. In contrast, the immunoassay technology is characterized by simple operation, rapidity and accuracy, and is more suitable for large-scale screening and on-site detection. The immunoassay technology is mainly based on the principle of antigen-antibody binding. At present, there are few antibody products against avian influenza virus on the market, and their performances vary.

[0008] Based on the above background, the present invention aims to provide a high-performance monoclonal antibody against avian influenza virus and its preparation method, so as to play an important role in the field of rapid diagnosis of avian influenza virus. Summary of the Invention

[0009] The present application provides an antibody against avian influenza virus, which provides an important raw material source for the detection of avian influenza virus, has good activity and sensitivity, can effectively detect different subtypes of H5N1 antigens and strains, and only binds to H5N1 antigens, without binding to avian influenza antigens of other subtypes, and has good detection specificity.

[0010] To achieve the above object, according to the first aspect of the present invention, there is provided an antibody against avian influenza virus, wherein the antibody comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence shown in any one of SEQ ID NO: 31 to SEQ ID NO: 35 and three complementary determining regions of a light chain variable region having an amino acid sequence shown in any one of SEQ ID NO: 36 to SEQ ID NO: 40.

[0011] To achieve the above object, according to the second aspect of the present invention, there is provided an antibody against avian influenza virus, and the complementary determining regions of the antibody include any one of (a) to (e):

[0012] (a) HCDR1 with an amino acid sequence shown in SEQ ID NO: 1 (SYAMG), HCDR2 with an amino acid sequence shown in SEQ ID NO: 2 (IINTAGSAYYASWAKG), HCDR3 with an amino acid sequence shown in SEQ ID NO: 3 (GAHSIDYTYFDI), and LCDR1 with an amino acid sequence shown in SEQ ID NO: 4 (QASQSISSYLA), LCDR2 with an amino acid sequence shown in SEQ ID NO: 5 (QASKLAS), LCDR3 with an amino acid sequence shown in SEQ ID NO: 6 (QSYYGTSGTASYNA);

[0013] (b) HCDR1 with the amino acid sequence shown in SEQ ID NO:7 (SNAMG), HCDR2 with the amino acid sequence shown in SEQ ID NO:8 (TITTSGTTYYASWAKG), HCDR3 with the amino acid sequence shown in SEQ ID NO:9 (PYIGSSWGYYFNI), and LCDR1 with the amino acid sequence shown in SEQ ID NO:10 (QASENIYSGLA), LCDR2 with the amino acid sequence shown in SEQ ID NO:11 (SASTLAS), LCDR3 with the amino acid sequence shown in SEQ ID NO:12 (LYGDYTISSAFA);

[0014] (c) HCDR1 with the amino acid sequence shown in SEQ ID NO:13 (GSWMN), HCDR2 with the amino acid sequence shown in SEQ ID NO:14 (RTYPGDGDSKYNGIFKG), HCDR3 with the amino acid sequence shown in SEQ ID NO:15 (GRIPYYFDS), and LCDR1 with the amino acid sequence shown in SEQ ID NO:16 (RASESVDNYGNSFMN), LCDR2 with the amino acid sequence shown in SEQ ID NO:17 (LASNLEA), LCDR3 with the amino acid sequence shown in SEQ ID NO:18 (QQNNEDPWT);

[0015] (d) HCDR1 with the amino acid sequence shown in SEQ ID NO:19 (DSWIS), HCDR2 with the amino acid sequence shown in SEQ ID NO:20 (RIFPGDGDSKYSGKFKG), HCDR3 with the amino acid sequence shown in SEQ ID NO:21 (GVLPWYFDV), and LCDR1 with the amino acid sequence shown in SEQ ID NO:22 (RASESVDNYGNSFMH), LCDR2 with the amino acid sequence shown in SEQ ID NO:23 (RASNLES), LCDR3 with the amino acid sequence shown in SEQ ID NO:24 (QQSNEDPFT); and

[0016] (e) HCDR1 with the amino acid sequence shown in SEQ ID NO:25 (SYNFH), HCDR2 with the amino acid sequence shown in SEQ ID NO:26 (CIYPGNGGTNYSQKFRG), HCDR3 with the amino acid sequence shown in SEQ ID NO:27 (SYGTSYVGAMDY), and LCDR1 with the amino acid sequence shown in SEQ ID NO:28 (RASESVEYSGISLLQ), LCDR2 with the amino acid sequence shown in SEQ ID NO:29 (AASNVES), LCDR3 with the amino acid sequence shown in SEQ ID NO:30 (QQSRKVPST).

[0017] To achieve the above object, according to the third aspect of the present invention, there is provided an antibody against avian influenza virus, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 31 to SEQ ID NO: 35, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 36 to SEQ ID NO: 40.

[0018] To achieve the above object, according to the fourth aspect of the present invention, there is provided an antibody against avian influenza virus, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 41, 42, 43, 44, 45, 56, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 46 to SEQ ID NO: 50.

[0019] To achieve the above object, according to the fifth aspect of the present invention, there is provided an antibody conjugate, wherein the antibody conjugate comprises the above antibody.

[0020] To achieve the above object, according to the sixth aspect of the present invention, there is provided a reagent or a kit, wherein the reagent or the kit comprises the above antibody or the above antibody conjugate.

[0021] To achieve the above object, according to the seventh aspect of the present invention, there is provided a use of the above antibody and antibody conjugate in the preparation of a product for detecting avian influenza virus.

[0022] To achieve the above object, the present invention also provides a nucleic acid molecule, a vector, a cell and a method for preparing the above antibody. Detailed Embodiments

[0023] In the first aspect, an embodiment of the present invention provides an antibody against avian influenza virus, wherein the antibody comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 31 to SEQ ID NO: 35 and three complementary determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 36 to SEQ ID NO: 40.

[0024] In an alternative embodiment, the complementary determining regions of the variable regions are defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.

[0025] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, provided that they exhibit the desired antigen-binding activity. An antigen-binding fragment of an antibody is a substance that contains the CDRs of the antibody and lacks some of the amino acids present in the full-length chains but is still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), and minimal recognition units of antibodies. Antigen-binding fragments of antibodies generally have the same binding specificity as the antibody from which they are derived. It will be readily understood by those skilled in the art from the content described in the present invention that antigen-binding fragments of antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Based on the disclosure of the structure of intact antibodies in the present invention, it is easy for those skilled in the art to obtain antigen-binding fragments of antibodies.

[0026] Antigen-binding fragments of antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0027] In the present invention, the terms "complementary determining region", "CDR", or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, which refer to regions containing one or more or even all of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the specific embodiments of the present invention, the CDRs refer to the highly variable regions of the heavy and light chains of an antibody.

[0028] In the present invention, the heavy-chain complementary determining regions are denoted as HCDR and include HCDR1, HCDR2, and HCDR3; the light-chain complementary determining regions are denoted as LCDR and include LCDR1, LCDR2, and LCDR3.

[0029] The methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). For the "Chothia definition", see Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results for specific residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literatures may vary slightly. Given the amino acid sequence of the variable region of a given antibody, those skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods not limited to those in Table 1 also fall within the scope of protection of the present disclosure.

[0030] Table 1: CDR Definitions 1

[0031] CDR Kabat AbM2 IMGT Chothia HCDR1 <![CDATA[H31~H35 3 > <![CDATA[H26~H35 3 > <![CDATA[H26~H33..5 5 > <![CDATA[H26~H32..34 4 > HCDR2 H50 - H65 H50 - H58 H51 - H57 H52 - H56 HCDR3 H95 - H102 H95 - H102 H93 - H102 H95 - H102 LCDR1 L24 - L34 L24 - L34 L27 - L32 L24 - L34 LCDR2 L50 - L56 L50 - L56 L50 - L51 L50 - L56 LCDR3 L89 - L97 L89 - L97 L89 - L97 L89 - L97

[0032] 1 The numbers for all CDR definitions in Table 1 are based on the Kabat numbering system (see below), and the amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number". Those of ordinary skill in the art can clearly map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, the "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0033] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDR defined by the "AbM" antibody modeling software of Oxford Molecular.

[0034] 3If neither H35A nor H35B exists, then HCDR1 ends at position 35; if only H35A exists, then HCDR1 ends at position 35A; if both H35A and H35B exist, then HCDR1 ends at position 35B.

[0035] 4 If neither H35A nor H35B exists, then HCDR1 ends at position 32; if only H35A exists, then HCDR1 ends at position 33; if both H35A and H35B exist, then HCDR1 ends at position 34.

[0036] 5 If neither H35A nor H35B exists, then HCDR1 ends at position 33; if only H35A exists, then HCDR1 ends at position 34; if both H35A and H35B exist, then HCDR1 ends at position 35.

[0037] According to an embodiment of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

[0038] In some alternative embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0039] In some alternative embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0040] In some alternative embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0041] In some alternative embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0042] In some alternative embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0043] In some alternative embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM, or Contact systems.

[0044] In a second aspect, an embodiment of the present invention provides an antibody against avian influenza virus, and the complementarity-determining regions of the aforementioned antibody include any one of (a) to (e):

[0045] (a) HCDR1 with the amino acid sequence shown in SEQ ID NO: 1 (SYAMG), HCDR2 with the amino acid sequence shown in SEQ ID NO: 2 (IINTAGSAYYASWAKG), HCDR3 with the amino acid sequence shown in SEQ ID NO: 3 (GAHSIDYTYFDI), and LCDR1 with the amino acid sequence shown in SEQ ID NO: 4 (QASQSISSYLA), LCDR2 with the amino acid sequence shown in SEQ ID NO: 5 (QASKLAS), and LCDR3 with the amino acid sequence shown in SEQ ID NO: 6 (QSYYGTSGTASYNA);

[0046] (b) HCDR1 with the amino acid sequence shown in SEQ ID NO: 7 (SNAMG), HCDR2 with the amino acid sequence shown in SEQ ID NO: 8 (TITTSGTTYYASWAKG), HCDR3 with the amino acid sequence shown in SEQ ID NO: 9 (PYIGSSWGYYFNI), and LCDR1 with the amino acid sequence shown in SEQ ID NO: 10 (QASENIYSGLA), LCDR2 with the amino acid sequence shown in SEQ ID NO: 11 (SASTLAS), and LCDR3 with the amino acid sequence shown in SEQ ID NO: 12 (LYGDYTISSAFA);

[0047] (c) HCDR1 with the amino acid sequence shown in SEQ ID NO: 13 (GSWMN), HCDR2 with the amino acid sequence shown in SEQ ID NO: 14 (RTYPGDGDSKYNGIFKG), HCDR3 with the amino acid sequence shown in SEQ ID NO: 15 (GRIPYYFDS), and LCDR1 with the amino acid sequence shown in SEQ ID NO: 16 (RASESVDNYGNSFMN), LCDR2 with the amino acid sequence shown in SEQ ID NO: 17 (LASNLEA), and LCDR3 with the amino acid sequence shown in SEQ ID NO: 18 (QQNNEDPWT);

[0048] (d) An HCDR1 with an amino acid sequence as shown in SEQ ID NO:19 (DSWIS), an HCDR2 with an amino acid sequence as shown in SEQ ID NO:20 (RIFPGDGDSKYSGKFKG), an HCDR3 with an amino acid sequence as shown in SEQ ID NO:21 (GVLPWYFDV), and an LCDR1 with an amino acid sequence as shown in SEQ ID NO:22 (RASESVDNYGNSFMH), an LCDR2 with an amino acid sequence as shown in SEQ ID NO:23 (RASNLES), an LCDR3 with an amino acid sequence as shown in SEQ ID NO:24 (QQSNEDPFT); and

[0049] (e) An HCDR1 with an amino acid sequence as shown in SEQ ID NO:25 (SYNFH), an HCDR2 with an amino acid sequence as shown in SEQ ID NO:26 (CIYPGNGGTNYSQKFRG), an HCDR3 with an amino acid sequence as shown in SEQ ID NO:27 (SYGTSYVGAMDY), and an LCDR1 with an amino acid sequence as shown in SEQ ID NO:28 (RASESVEYSGISLLQ), an LCDR2 with an amino acid sequence as shown in SEQ ID NO:29 (AASNVES), an LCDR3 with an amino acid sequence as shown in SEQ ID NO:30 (QQSRKVPST).

[0050] According to an embodiment of the present invention, the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0051] In an alternative embodiment, the antibodies described in the first aspect and the second aspect further include a framework region.

[0052] In an alternative embodiment, the antibodies described in the first aspect and the second aspect further have framework regions as shown in HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0053] In an alternative embodiment, the heavy chain variable region and the light chain variable region of the antibodies described in the first aspect and the second aspect are selected from any one of (a') to (e'):

[0054] (a') A heavy chain variable region with an amino acid sequence having at least 80% identity to SEQ ID NO:31, a light chain variable region with an amino acid sequence having at least 80% identity to SEQ ID NO:36, and including the complementarity determining regions shown in (a) of the second aspect;

[0055] (b’) A heavy chain variable region with an amino acid sequence having at least 80% identity with SEQ ID NO: 32, a light chain variable region with an amino acid sequence having at least 80% identity with SEQ ID NO: 37, and comprising the complementarity determining regions shown in (b) of the second aspect;

[0056] (c’) A heavy chain variable region with an amino acid sequence having at least 80% identity with SEQ ID NO: 33, a light chain variable region with an amino acid sequence having at least 80% identity with SEQ ID NO: 38, and comprising the complementarity determining regions shown in (c) of the second aspect;

[0057] (d’) A heavy chain variable region with an amino acid sequence having at least 80% identity with SEQ ID NO: 34, a light chain variable region with an amino acid sequence having at least 80% identity with SEQ ID NO: 39, and comprising the complementarity determining regions shown in (d) of the second aspect; and

[0058] (e’) A heavy chain variable region with an amino acid sequence having at least 80% identity with SEQ ID NO: 35, a light chain variable region with an amino acid sequence having at least 80% identity with SEQ ID NO: 40, and comprising the complementarity determining regions shown in (e) of the second aspect.

[0059] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the regions other than the complementarity determining regions CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein, the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0060] In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: HFR1 - HCDR1 - HFR2 - HCDR2 - HFR3 - HCDR3 - HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: LFR1 - LCDR1 - LFR2 - LCDR2 - LFR3 - LCDR3 - LFR4.

[0061] In the present invention, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned. Alignment of the amino acid sequence identity percentage can be carried out in various ways in the art, such as software known in the art such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW or CLUSTAL OMEGA, etc.

[0062] In other embodiments, the amino acid sequences of each framework region of the anti-avian influenza virus antibody provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the corresponding framework regions described above.

[0063] In a third aspect, an embodiment of the present invention provides an anti-avian influenza virus antibody, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NOs: 31 to 35, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NOs: 36 to 40.

[0064] In an alternative embodiment, the combination of the above heavy chain variable region and light chain variable region is selected from any combination of (A) to (E):

[0065] (A) A heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 31, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 36;

[0066] (B) A heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 32, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 37;

[0067] (C) A heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 33, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 38;

[0068] (D) A heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 34, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 39; and

[0069] (E) A heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 35, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 40.

[0070] In an alternative embodiment, the antibody according to the first, second or third aspect further comprises a constant region.

[0071] In an alternative embodiment, the constant region includes a heavy chain constant region and a light chain constant region.

[0072] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

[0073] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0074] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0075] In an alternative embodiment, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0076] In an alternative embodiment, the species origin of the constant region is bovine, equine, porcine, ovine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

[0077] In an alternative embodiment, the species origin of the constant region is rabbit.

[0078] In an alternative embodiment, the species origin of the constant region is mouse.

[0079] In this article, the division of variable region and constant region sequences refers to the IMGT division method. See Lefranc, the international ImMunoGeneTics database. Nucl. Acids Res., 29(1):207 - 209(2001). DOI:10.1093 / nar / 29.1.207. PMID:11125093. and Martinez-Jean C. and Bosc N. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: housemouse (Mus musculus) IGHC, IMGT Repertoire. the internationalImMunoGenetics information http: / / www.imgt.org.Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. Or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGLC, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org .Created: 16 / 03 / 2011. Version: 17 / 01 / 2020.. There will be some amino acid differences between the variable regions divided by different methods and the C-terminus of the variable region or the N-terminus of the constant region divided by IMGT. The variable regions or constant regions divided by other methods well-known in the art are also within the protection scope of the present invention.

[0080] In an alternative embodiment, the above-mentioned constant region is selected from any one of (F) to (I):

[0081] (F) CH with the amino acid sequence shown in SEQ ID NO: 51; and CL with the amino acid sequence shown in SEQ ID NO: 52; or an amino acid sequence having at least 80% identity with each of the constant regions;

[0082] (G) CH with the amino acid sequence shown in SEQ ID NO: 53; and CL with the amino acid sequence shown in SEQ ID NO: 55; or an amino acid sequence having at least 80% identity with each of the constant regions;

[0083] (H) CH with the amino acid sequence shown in SEQ ID NO: 54; and CL with the amino acid sequence shown in SEQ ID NO: 55; or an amino acid sequence having at least 80% identity with each of the constant regions; and

[0084] (I) CH with the amino acid sequence shown in SEQ ID NO: 57; and CL with the amino acid sequence shown in SEQ ID NO: 52; or an amino acid sequence having at least 80% identity with each of the constant regions.

[0085] In other embodiments, the above-mentioned constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the constant region.

[0086] In an alternative embodiment, the above-mentioned antibody includes any one of F(ab)2, F(ab’) 2 , Fab’, Fab, Fv and scFv.

[0087] In a fourth aspect, the present invention provides an antibody against avian influenza virus, including a heavy chain and / or a light chain, wherein the amino acid sequence of the aforementioned heavy chain is as shown in any one of SEQ ID NO:41, 42, 43, 44, 45, 56, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO:46 to SEQ ID NO:50.

[0088] In an alternative embodiment, the combination of the above-mentioned heavy chain and light chain is selected from any combination of (A’) to (F’):

[0089] (A’) a heavy chain with the amino acid sequence as shown in SEQ ID NO:41, and a light chain with the amino acid sequence as shown in SEQ ID NO:46;

[0090] (B’) a heavy chain with the amino acid sequence as shown in SEQ ID NO:42, and a light chain with the amino acid sequence as shown in SEQ ID NO:47;

[0091] (C’) a heavy chain with the amino acid sequence as shown in SEQ ID NO:43, and a light chain with the amino acid sequence as shown in SEQ ID NO:48;

[0092] (D’) a heavy chain with the amino acid sequence as shown in SEQ ID NO:44, and a light chain with the amino acid sequence as shown in SEQ ID NO:49;

[0093] (E’) a heavy chain with the amino acid sequence as shown in SEQ ID NO:45, and a light chain with the amino acid sequence as shown in SEQ ID NO:50; and

[0094] (F’) a heavy chain with the amino acid sequence as shown in SEQ ID NO:56, and a light chain with the amino acid sequence as shown in SEQ ID NO:46.

[0095] In a fifth aspect, the present invention provides an antibody conjugate, and the aforementioned antibody conjugate includes the above-mentioned antibody.

[0096] In an alternative embodiment, the above-mentioned antibody conjugate includes biotin or a biotin derivative.

[0097] In an alternative embodiment, the above-mentioned antibody conjugate further includes a label or a purification tag.

[0098] In an alternative embodiment, the above-mentioned marker refers to a class of substances with characteristics such as luminescence, color development, radioactivity, etc., which can be directly observed by the naked eye or detected or detected by an instrument. Through these characteristics, qualitative or quantitative detection of the corresponding target can be achieved.

[0099] In an alternative embodiment, the above-mentioned markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based markers.

[0100] In actual use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. No matter which marker is used, it falls within the protection scope of the present invention.

[0101] In an alternative embodiment, the above-mentioned fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0102] In an alternative embodiment, the above-mentioned enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0103] In an alternative embodiment, the above-mentioned radioisotopes include, but are not limited to 212 Bi, 131 I, 111 In, 90 Y, 186 Re, 211 At, 125 I, 188 Re, 153 Sm, 213 Bi, 32 P, 94 mTc, 99mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.

[0104] In alternative embodiments, the above chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, copepod luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetane and its derivatives, rhodamine and its derivatives, and peroxyoxalate and its derivatives.

[0105] In alternative embodiments, the above nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0106] In alternative embodiments, the above colloids include, but are not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0107] In alternative embodiments, the above colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0108] In alternative embodiments, the above antibody conjugate further includes a solid-phase carrier conjugated to the above antibody.

[0109] In alternative embodiments, the above solid-phase carrier is selected from microspheres, plates, and membranes.

[0110] In alternative embodiments, the above solid-phase carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microtiter plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

[0111] In a sixth aspect, the present invention provides a reagent or a kit, and the aforementioned reagent or kit includes the above antibody or the above antibody conjugate.

[0112] As described above, the antibodies in some embodiments or examples of the present invention can effectively bind to avian influenza virus. Therefore, reagents or kits containing the above-mentioned avian influenza virus antibodies can effectively detect avian influenza virus qualitatively or quantitatively. Using the reagents or kits provided by the present invention, for example, they can be used in immunoblotting, immunoprecipitation and other detections involving the specific binding performance of avian influenza virus and its antibodies. As described above, the antibodies in some embodiments or examples of the present invention have higher binding activity and specificity with avian influenza virus. Therefore, the reagents or kits containing the above-mentioned antibodies have higher detection sensitivity or specificity.

[0113] In a seventh aspect, the present invention provides a method for detecting avian influenza virus, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with avian influenza virus in a test sample under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the aforementioned immune complex, the presence of which indicates the presence of the above-mentioned antigen in the test sample.

[0114] In an alternative embodiment, the above-mentioned immune complex further comprises a second antibody that binds to the above-mentioned antibody.

[0115] In an alternative embodiment, the above-mentioned immune complex further comprises a second antibody that binds to avian influenza virus.

[0116] In an eighth aspect, the present invention provides the use of the above-mentioned anti-avian influenza virus antibody and antibody conjugate in the preparation of products for detecting avian influenza virus.

[0117] It should be noted that the products of the present invention include but are not limited to reagents, kits, test strips or reagent plates.

[0118] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0119] In a tenth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0120] In an eleventh aspect, the present invention provides a cell containing the above-mentioned vector.

[0121] In a twelfth aspect, the present invention provides a method for preparing an anti-avian influenza virus antibody, which comprises: culturing the cells as described above.

[0122] In the present invention, the term "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, and nucleic acid molecules include ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemical or biochemical modifications, unnatural or derivatized nucleobases. When a nucleic acid molecule encodes a protein or polypeptide, the encoding optionally encodes the sense strand or the antisense strand. Nucleic acid molecules can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably.

[0123] In the present invention, the term "vector" refers to a vehicle into which a genetic element (such as the aforementioned nucleic acid molecule) can be operably inserted and which enables the genetic element to be expressed, for example, to produce a protein, RNA or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce or transfect host cells so that the genetic elements they carry are expressed in the host cells. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phages or M13 phages, and animal viruses. Vectors can contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, vectors can also contain an origin of replication. Vectors can also include components that assist their entry into cells, including, but not limited to, viral particles, liposomes or protein coats. Vectors can be expression vectors or cloning vectors.

[0124] In the present invention, the term "recombinant cell" refers to a cell that can or has been introduced with an exogenous polynucleotide and / or a vector. The exogenous polynucleotide can be integrated or not integrated into the genome of the "recombinant cell". When the recombinant cell contains a vector, the vector can be introduced into mammalian cells to construct recombinant cells, and then these recombinant cells can be used to express the antibodies or antigen-binding fragments provided by the present invention. By culturing the recombinant cells, the corresponding antibodies can be obtained. Available mammalian cells can be CHO cells, etc.

[0125] Based on the disclosure of the amino acid sequence of the anti-avian influenza virus antibody in the present invention, those skilled in the art can easily conceive of preparing the anti-avian influenza virus antibody by using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression). For example, the antibody can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody described in any one of the above. This is easily achievable for those skilled in the art. Based on this, regardless of the technique used to prepare the anti-avian influenza virus antibody of the present invention, it falls within the protection scope of the present invention.

[0126] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0127] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise noted, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

[0128] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", 2nd Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Weir and C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller and M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1991), each of which is hereby expressly incorporated by reference.

[0129] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0130] Example 1 Antibody Discovery of Monoclonal Antibodies

[0131] Method 1:

[0132] 1. Animal Immunization

[0133] Mix the H5N1 HA protein (from PhyGenesys) with an equal volume of Freund's complete adjuvant to obtain an oily emulsion. Subcutaneously inject the BALB / c mice at multiple points with a dose of 0.2 ml per mouse. After 14 days, immunize the mice intraperitoneally with the same antigen and adjuvant. Immunize until the fourth injection, collect tail blood for titer detection, and the titer meets the fusion requirement. Three days before fusion, mix the same dose of antigen with an equal volume of 0.9% sodium chloride injection and inject intraperitoneally for booster immunization.

[0134] 2. Preparation of hybridoma cell line

[0135] On the third day after the booster immunization of the mice, remove the spleens under sterile conditions. Mix the mouse tumor cells and immunized spleen cells at a cell number ratio of 1:10, fuse, and culture. On the sixth day of culture, change the HT culture medium twice. On the seventh day after fusion, collect the cell supernatant for antibody detection to screen for hybridoma cell strains secreting specific antibodies. A total of 3 strains were named 12F8, 10B6, and 6H4.

[0136] 3. Antibody gene sequencing of hybridoma cells

[0137] Extract the RNA of 12F8, 10B6, and 6H4 hybridoma cells, reverse transcribe it into cDNA, then perform PCR amplification of the antibody gene fragment. Next, insert and ligate the antibody gene fragment into the sequencing T vector (purchased from Takara). Finally, perform sequencing of the antibody gene to obtain the gene sequence of the antibody variable region.

[0138] Perform eukaryotic recombinant expression on the anti-H5N1 rabbit monoclonal antibody sequence obtained by the above method.

[0139] Method 2:

[0140] 1. Immunize animals

[0141] Use the emulsified preparation of H5N1 HA protein prepared with incomplete Freund's adjuvant to stimulate the immune response of New Zealand white rabbits at 4 - 6 weeks old by subcutaneous injection. Collect pre-immune and post-immune sera at 0, 14, 28, 42, and 69 days respectively for serum titer detection; select rabbits with qualified titers, surgically remove the rabbit spleens, and prepare spleen cell suspensions. Then, isolate fresh single spleen cells and culture them overnight in B cell medium.

[0142] 2. Obtain specific single B cells

[0143] 1) Dilute the spleen cells with PBS solution containing 2 - 3% fetal bovine serum and 1 mM EDTA to obtain a fresh cell suspension.

[0144] 2) Single B cells were sorted for H5N1 HA antigen specificity using a Sony MA900 flow sorter (Sony Biotechnology, Japan) and placed in each well of a 96-well plate.

[0145] 3) Primary B cells with H5N1 HA specificity were added to B cell medium and then cultured at 37 °C and 5.5% CO2 for 7 - 10 days.

[0146] 4) At the end of the primary B cell culture, the B cell culture supernatant was screened and identified by ELISA. Finally, 2 B cell positive clones with superior detection performance against H5N1 were selected and named 8J13 and 10D13 respectively.

[0147] 3. Sequencing of B cell antibody genes:

[0148] RNA was extracted from the 8J13 and 10D13 positive clone cells and reverse transcribed into cDNA. Then, PCR amplification of the antibody gene fragments was carried out. Next, the antibody gene fragments were inserted and ligated into a sequencing T vector (purchased from Takara). Finally, sequencing of the antibody gene was performed to obtain the gene sequences of the antibody variable regions.

[0149] The anti - H5N1 rabbit monoclonal antibody sequences obtained by the above method were subjected to eukaryotic recombinant expression.

[0150] Example 2 Preparation of Monoclonal Antibodies

[0151] 1. Construction of recombinant antibody expression plasmids

[0152] pcDNA TM 3.4 The vector pcDNA3.4A is the constructed recombinant antibody eukaryotic expression vector. This expression vector has introduced multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI, and is named the pcDNA3.4A expression vector, hereinafter simply referred to as the 3.4A expression vector. According to the sequencing results of the variable regions of the above antibodies 12F8, 10B6, 6H4, 8J13, and 10D13, gene - specific primers for the light - chain variable region and heavy - chain variable region of the antibody were designed, with HindIII and EcoRI enzyme digestion sites and protective bases at both ends. The light - chain gene fragment and heavy - chain gene fragment were amplified by PCR amplification.

[0153] The heavy - chain and light - chain gene fragments were respectively double - digested with HindIII / EcoRI, and the 3.4A vector was double - digested with HindIII / EcoRI. After the fragments and the vector were purified and recovered, the heavy - chain gene and light - chain gene were respectively ligated into the 3.4A expression vector to obtain recombinant expression plasmids for the heavy - chain and light - chain respectively.

[0154] 2. Recombinant antibody production

[0155] 2.1 Recombinant cell antibody expression

[0156] Resuscitate HEK293 cells in advance, passage and culture them in a 200 mL system until the cell density reaches 3 - 5×10 6 cells / mL. Select antibodies and cells with a cell density reaching the required concentration and a cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 3.3×10 6 cells / mL as the cell dilution. Prepare plasmid DNA and transfection reagent dilutions with the medium respectively. Add the transfection reagent dilution to the plasmid DNA dilution, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell dilution within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in an incubator at 35°C for culture at a rotation speed of 120 rmp and a CO 2 content of 8%. After 13 days, centrifuge to collect the samples and detect the cell supernatant by ELISA.

[0157] 2.1.1 Indirect ELISA supernatant detection

[0158] 1) Coating: Coat the H5N1 antigen (from Affinity Biologicals) at a concentration of 0.5 μg / mL and incubate overnight at 4°C.

[0159] 2) Wash twice with PBST, pat dry, block with 20% bovine serum at 120 μL / well and incubate at 37°C for 1 h, then pat dry.

[0160] 3) Dilute the supernatants of 8J13 and 10D13 cells 500-fold, 2000-fold, 2000-fold, and 10000-fold respectively with 20% bovine serum, and add 100 μL / well to the ELISA plate in step 2); dilute the supernatants of 12F8, 10B6, and 6H4 cells with 20% bovine serum, perform serial dilutions starting from 240-fold to 15360-fold, and add 100 μL / well to the ELISA plate in step 2); incubate at 37°C for 30 min.

[0161] 4) Wash 5 times with PBST, pat dry, add goat anti-rabbit IgG-HRP (diluted 5000-fold with 1% casein) to the ELISA plates corresponding to 8J13 and 10D13, and add goat anti-mouse IgM-HRP (diluted 5000-fold with 1% casein) to the ELISA plates corresponding to 12F8, 10B6, and 6H4, then incubate at 37°C for 30 min.

[0162] 5) Wash 5 times with PBST, pat dry, add 50 μL of Solution A and 50 μL of Solution B respectively, incubate in the dark for 10 min, add 50 μL of the stop solution, read with an ELISA reader. The detection results of 8J13 and 10D13 are shown in Table 3, and the detection results of 12F8, 10B6, and 6H4 are shown in Table 4. The results show that antibodies 8J13, 10D13, 12F8, 10B6, and 6H4 can all effectively bind to the H5N1 antigen.

[0163] Table 3: Indirect ELISA Cell Supernatant Binding Activity Data - 1

[0164] Dilution factor Original concentration 500 - fold 2000 - fold 10000 - fold 10D13 2.142 2.155 2.226 1.786 8J13 2.026 2.169 2.134 2.008

[0165] Table 4: Indirect ELISA Cell Supernatant Binding Activity Data - 2

[0166] Dilution factor 240 480 960 1920 3840 7680 15360 12F8 1.991 1.953 1.642 1.229 0.732 0.365 0.256 10B6 2.178 1.864 1.134 0.632 0.362 0.2 0.107 6H4 2.017 2.034 1.771 1.225 0.71 0.411 0.225

[0167] 2.2 Antibody Supernatant Purification

[0168] Centrifuge the recombinant expressed antibody supernatant and purify it by affinity chromatography using a protein A affinity column to obtain the purified antibody. The obtained antibodies are named 10D13 Rmb, 8J13 Rmb, 12F8 Rmb, 10B6 Rmb, and 6H4 Rmb. The amino acid sequences of the heavy chains and light chains of each antibody are shown in Table 5.

[0169] Table 5: Antibody Sequences

[0170]

[0171]

[0172] Example 3 Performance Detection of Antibodies

[0173] 1. Antibody Labeling Process

[0174] (1) Adjust the labeling pH: Take 10 mL of 4 / 10,000 colloidal gold, centrifuge, add 150 μL of 0.2M K 2 CO 3 150 μL, stir and mix well for 2 min;

[0175] (2) Couple the antibody: Add 200 μg of antibodies 6H4 Rmb, 8J13 Rmb, and 10D13 Rmb respectively, and stir and react for 15 min;

[0176] (3) Block: Add 10% (mass - volume ratio) BSA and stir and react for 15 min;

[0177] (4) Centrifuge and store: 10000 rpm / 7 min / room temperature, remove the supernatant, resuspend with the gold - labeled complex solution to 1 mL, and store at 4°C for later use;

[0178] 2. Coating

[0179] (1) Assemble the nitrocellulose membrane and the colloidal gold PVC bottom plate for later use;

[0180] (2) Dilute antibodies 12F8 Rmb, 10B6 Rmb, and 8J13 Rmb to 1.0 - 2.0 mg / mL respectively. Use a gold-spraying and membrane-drawing instrument to draw lines evenly on the NC membrane, and then place it in an incubator at 50 °C for drying for at least 4 hours. Assemble, cut into strips, and perform sample addition and detection.

[0181] 3. Detection

[0182] (1) Samples:

[0183] Sample 1: Recombinant antigens of different concentrations of H5N1;

[0184] Sample 2: H5N1 virus strains from different sources (from NIBSC);

[0185] Sample 3: Avian influenza viruses of different subtypes;

[0186] (2) Detection method: Colloidal gold detection. Observe the depth of the detection line with the naked eye to determine the result. According to the depth of the color of the displayed band, the activity of the antigen-antibody binding in the sample can be indicated. Compare the color of the T line strip showing color reaction of the colloidal gold test strip with the standard color card, select the closest color, and mark the activity of the product with the number of the color number corresponding to this color.

[0187] The smaller the number, the stronger the color development and the higher the activity; the higher the number, the weaker the color development and the lower the activity; the number with a "+" indicates slightly stronger, the number with a "-" indicates slightly weaker, and "B" represents negative.

[0188] (3) Detection results

[0189] The detection results of each antibody pair are shown in Table 6. The results show that (1) the pairs composed of antibodies 10D13 Rmb, 8J13 Rmb, 12F8 Rmb, 10B6 Rmb, and 6H4 Rmb can all effectively detect H5N1, and all only bind to the H5N1 antigen and do not bind to other subtype avian influenza antigens, with good detection specificity.

[0190] Table 6: Detection results of the colloidal gold platform

[0191]

[0192] Example 4 Construction, Preparation and Performance Detection of IgG / IgM Hybrid Recombinant Antibodies

[0193] 1. Construction and Preparation of IgG / IgM Hybrid Recombinant Antibodies

[0194] The constant region of the 8J13 Rmb antibody was modified to construct an IgG / IgM hybrid recombinant antibody.

[0195] 1.1 Construction of the expression plasmid of the IgG / IgM hybrid recombinant antibody

[0196] The heavy chain variable region segment encoding 8J13 Rmb was spliced together with the sequence encoding the IgM constant region by overlap extension PCR. The polynucleotide obtained by PCR amplification was digested with HindIII / EcoRI and then ligated into the 3.4A expression vector. Since the multimeric property of IgM depends on the heavy chain, the light chain expression plasmid of 8J13 Rmb was used.

[0197] 1.2 Expression and preparation of the IgG / IgM hybrid recombinant antibody

[0198] The heavy chain and light chain expression plasmids of the IgG / IgM hybrid recombinant antibody were diluted at a ratio of 1:1 and mixed with CHO cells. After electroporation, the cells were cultured and pressurized with MSX for about 25 days. The cell viability was recorded and observed. The cell line with a high antibody concentration was selected for scale-up culture and placed in a 37 °C constant temperature incubator with a rotation speed of 120 rmp and a CO 2 content of 8%. After 13 days, the samples were collected by centrifugation to obtain the cell line of the IgG / IgM hybrid recombinant antibody, which was named 8J13A. The cell supernatant of 8J13A was diluted 100-fold, 200-fold, and 400-fold with 20% bovine serum respectively for ELISA detection. The detection method was the same as 2.1.1. The detection results are shown in Table 7, indicating that 8J13A can effectively bind to the H5N1 antigen.

[0199] Table 7: Binding activity data of cell supernatant by indirect ELISA method - 3

[0200] Dilution factor Original concentration 100 - fold 200 - fold 400 - fold 8J13A 2.155 1.907 1.315 0.756

[0201] 2. Expression of recombinant antibody

[0202] The IgG / IgM hybrid recombinant antibody expression supernatant was purified in two steps using two fillers, captoL (cytiva) and CHT (Bio-Rad), to obtain the recombinant antibody, which was named 8J13A Rmb. The heavy chain amino acids of 8J13A Rmb are shown in SEQ ID NO:56, and the light chain amino acids are shown in SEQ ID NO:46.

[0203] 3. Performance detection of the antibody

[0204] The paired reagents composed of antibody 8J13A Rmb were used to detect different samples. The detection method was the same as that in Example 3. The samples included H5N1 recombinant antigens of different subtypes, H5N1 strains from different sources (from NIBSC), and avian influenza viruses of different subtypes. The detection results are shown in Table 8. The results showed that the pairs composed of 8J13A Rmb had stronger detection ability for recombinant antigens of different H5N1 subtypes and different H5N1 strains, and all only bound to H5N1 antigens and did not bind to avian influenza antigens of other subtypes, with good detection specificity.

[0205] Table 8: Detection Results of Colloidal Gold Platform - 2

[0206]

[0207]

[0208] Some of the amino acid sequences involved in this application are shown in Table 9:

[0209] Table 9: Amino Acid Sequences

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An antibody against avian influenza virus, characterized in that: The antibody comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 31 to SEQ ID NO: 35 and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 36 to SEQ ID NO: 40; Optionally, the complementarity determining regions of the variable regions are defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.

2. The antibody according to claim 1, characterized in that The complementary determining region of the antibody includes any one of (a) to (e): (a) an amino acid sequence of HCDR1 represented by SEQ ID NO: 1 (SYAMG), HCDR2 represented by SEQ ID NO: 2 (IINTAGSAYYASWAKG), HCDR3 represented by SEQ ID NO: 3 (GAHSIDYTYFDI), and an amino acid sequence of LCDR1 represented by SEQ ID NO: 4 (QASQSISSYLA), LCDR2 represented by SEQ ID NO: 5 (QASKLAS), LCDR3 represented by SEQ ID NO: 6 (QSYYGTSGTASYNA); (b) an amino acid sequence of HCDR1 as shown in SEQ ID NO:7 (SNAMG), HCDR2 as shown in SEQ ID NO:8 (TITTSGTTYYASWAKG), HCDR3 as shown in SEQ ID NO:9 (PYIGSSWGYYFNI), and an amino acid sequence of LCDR1 as shown in SEQ ID NO:10 (QASENIYSGLA), LCDR2 as shown in SEQ ID NO:11 (SASTLAS), LCDR3 as shown in SEQ ID NO:12 (LYGDYTISSAFA); (c) an amino acid sequence of HCDR1 as shown in SEQ ID NO: 13 (GSWMN), HCDR2 as shown in SEQ ID NO: 14 (RTYPGDGDSKYNGIFKG), HCDR3 as shown in SEQ ID NO: 15 (GRIPYYFDS), and an amino acid sequence of LCDR1 as shown in SEQ ID NO: 16 (RASESVDNYGNSFMN), LCDR2 as shown in SEQ ID NO: 17 (LASNLEA), and LCDR3 as shown in SEQ ID NO: 18 (QQNNEDPWT); (d) an amino acid sequence of HCDR1 as shown in SEQ ID NO: 19 (DSWIS), HCDR2 as shown in SEQ ID NO: 20 (RIFPGDGDSKYSGKFKG), HCDR3 as shown in SEQ ID NO: 21 (GVLPWYFDV), and an amino acid sequence of LCDR1 as shown in SEQ ID NO: 22 (RASESVDNYGNSFMH), LCDR2 as shown in SEQ ID NO: 23 (RASNLES), LCDR3 as shown in SEQ ID NO: 24 (QQSNEDPFT); and (e) an amino acid sequence of HCDR1 as shown in SEQ ID NO:25 (SYNFH), HCDR2 as shown in SEQ ID NO:26 (CIYPGNGGTNYSQKFRG), and HCDR3 as shown in SEQ ID NO:27 (SYGTSYVGAMDY), and an amino acid sequence of LCDR1 as shown in SEQ ID NO:28 (RASESVEYSGISLLQ), LCDR2 as shown in SEQ ID NO:29 (AASNVES), and LCDR3 as shown in SEQ ID NO:30 (QQSRKVPST).

3. The antibody according to any one of claims 1 to 2, characterized in that The antibody further comprises a framework region; Optionally, the heavy chain variable region and light chain variable region of the antibody are selected from any one of (a') to (e'): (a') a heavy chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO:31, and a light chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO:36, and including the complementarity determining region shown in (a) of claim 2; (b') a heavy chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO:32, and a light chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO:37, and including the complementarity determining region shown in (b) of claim 2; (c') a heavy chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO: 33, and a light chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO: 38, and comprising the complementarity determining region shown in (c) of claim 2; (d') a heavy chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO: 34, and a light chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO: 39, and including the complementarity determining region shown in (d) of claim 2; and (e') a heavy chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO:35, and a light chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO:40, and including the complementarity determining region shown in (e) of claim 2.

4. An anti-avian influenza virus antibody comprising a heavy chain variable region and a light chain variable region, characterized in that: The heavy chain variable region amino acid sequence is shown in any one of SEQ ID NO:31 to SEQ ID NO:35; and the light chain variable region amino acid sequence is shown in any one of SEQ ID NO:36 to SEQ ID NO:40; Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any combination of (A) to (E): (A) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:31, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:36; (B) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:32, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:37; (C) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:33, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:38; (D) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:34, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:39; and (E) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:35, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

40.

5. The antibody according to any one of claims 1 to 4, characterized in that The antibody further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and a light chain constant region; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the light chain constant region comprises a light chain constant region selected from a κ-type or a λ-type; Optionally, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is rabbit; Optionally, the species origin of the constant region is mouse; Optionally, the constant region is selected from any one of (F) to (I): (F) CH with an amino acid sequence as shown in SEQ ID NO:51; and CL with an amino acid sequence as shown in SEQ ID NO:52; or an amino acid sequence having at least 80% identity with each of the constant regions; (G) CH with an amino acid sequence as shown in SEQ ID NO: 53; and CL with an amino acid sequence as shown in SEQ ID NO: 55; or an amino acid sequence having at least 80% identity with each of the constant regions; (H) CH with an amino acid sequence as shown in SEQ ID NO: 54; and CL with an amino acid sequence as shown in SEQ ID NO: 55; or an amino acid sequence having at least 80% identity with each of said constant regions; and (I) CH with an amino acid sequence as shown in SEQ ID NO: 57; and CL with an amino acid sequence as shown in SEQ ID NO: 52; or amino acid sequences having at least 80% identity with said respective constant regions.

6. An anti-avian influenza virus antibody, comprising a heavy chain and a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in any one of SEQ ID NO:41, 42, 43, 44, 45, and 56; the amino acid sequence of the light chain is shown in any one of SEQ ID NO:46 to SEQ ID NO:

50.

7. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody according to any one of claims 1 to 6; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a marker or purification tag coupled to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody.

8. A reagent or a kit, characterized in that: The reagent or kit comprises the antibody according to any one of claims 1 to 6 or the antibody conjugate according to claim 7.

9. Use of the antibody according to any one of claims 1 to 6 or the antibody conjugate according to claim 7 in the preparation of a product for detecting avian influenza virus; Optionally, the use includes: a) contacting the antibody according to any one of claims 1 to 6, the antibody conjugate according to claim 7, or the reagent or kit according to claim 8 with the avian influenza virus in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to the avian influenza virus.

10. A nucleic acid molecule, a vector, a cell or a method for preparing the antibody according to any one of claims 1 to 6, wherein the nucleic acid molecule encodes the antibody according to any one of claims 1 to 6; the vector contains a nucleic acid molecule encoding the antibody according to any one of claims 1 to 6; the cell contains the above-mentioned nucleic acid molecule or vector; the method comprises culturing the above-mentioned cell.

Images